The effective diagnosis and treatment of life-threatening fungal infections in humans is a major unmet clinical challenge, and medical mycology is the research discipline that addresses this need. Well-annotated genomic sequences of the major human fungal pathogens have recently been published, creating an opportunity to revolutionize medical mycology through the application of systematic genome-wide approaches. Just as the availability of genome-wide knockout collections has been instrumental in dissecting the biology of model fungi such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, the generation of analogous complete gene deletion collections in pathogenic fungi would create experimental tools to systematically elucidate the basis of fungal virulence in the mammalian host and to develop drugs against them. Cryptococcus neoformans is an encapsulated budding yeast that is the most common cause of fungal meningitis. There are an estimated 1,000,000 cases that result in ~600,000 deaths each year. It is estimated to cause one-third of deaths in AIDS patients worldwide. Using optimized particle bombardment methods for gene targeting, our laboratory demonstrated the feasibility of creating large numbers of bar-coded gene deletions in this organism. Specifically, we reported previously the construction of 1201 gene deletion strains. This collection has been made available without restriction to the community. We demonstrated the utility of this collection by using it in systematic screens of infectivity in experimental mice, expression of known virulence factors, mechanisms of hypoxic adaptation, and mechanisms of phagocytosis-inhibition. However, the impact of this collection is limited to the fraction of genes covered -- one-fifth of predicted gens. Objectives: We propose to generate and distribute a complete collection of knockout mutants for Cryptococcus neoformans. Knockouts will contain features to facilitate functional genomic studies. We further propose to obtain a series of reference phenotypes via quantitative screens of the library for defects in infectivity in vivo and the expression of the major virulence factorsin vitro. The proposed efort will be coordinated with those of others that aim to identify the essential genes of C. neoformans. Impact: The proposed work will transform medical mycology by providing the first full description of pathogen genes required for fitness during infection, a ull accounting of genes required for production of known virulence factors, and a permanent freely-available, nonredundant null mutant strain resource that will dramatically accelerate research in the field as a whole. PUBLIC HEALTH RELEVANCE: The diagnosis and treatment of serious fungal infections of humans is a major unmet clinical challenge. Cryptococcus neoformans is among the most important fungal pathogens, accounting for approximately one-third of worldwide deaths from HIV/AIDS. This study will generate unique resources that will facilitate a comprehensive understanding of how this pathogen causes disease and responds to drugs thereby providing new foundation for the development of much-needed therapeutics.